Clostridium difficile infection (CDI) has grown to be the most prevalent cause of hospital acquired infection in the United States. Susceptibility to CDI is induced by recent antibiotic exposure, which is known to alter the structure of the gut microbiome and to affect the availability of growth nutrients in the gut. We hypothesized that C. difficile is a generalist that adapts its physiology to the nutrients available within the gut. We orally challenged C57BL/6 mice that previously received one of three antibiotics with C. difficile and demonstrated that it was able to colonize the cecum within 18 hours of infection. However, levels of both spore and toxin production, which are known to be affected by nutrient availability, varied between each antibiotic treatment group. To more closely investigate the specific responses of C. difficile as it colonized the cecum, we performed in vivo transcriptional analysis of C. difficile from cecal content of infected mice. This approach revealed variation in expression of genes that drive life-cycle switches as well as metabolic pathways associated with catabolizing a variety of carbon sources such as carbohydrates, amino acids, and amino sugars. To assess which substrates C. difficile was most likely exploiting in each antibiotic-perturbed microbiome, we developed a novel metabolite scoring algorithm within the genome-scale bipartite metabolic network of C. difficile that incorporated both network topology and transcript abundance to infer the likelihood that a given metabolite was acquired from the environment. Applying this approach, we found that C. difficile indeed occupies alternative nutrient niches across each antibiotic-perturbed microbiome and that the highlighted metabolites support significant growth, in vitro. Results from this analysis support the hypothesis that consumption of N-acetyl-D-glucosamine and Stickland fermentation substrates are central components of C. difficile's metabolic strategy and pathogenesis. This work has implications for elucidating specifics of the nutrient niche of C. difficile during infection and may lead to the discovery of targeted measures to prevent C. difficile colonization including potential pre- or probiotic therapies.